Inorganic beads with hierarchical pore structures

ABSTRACT

Disclosed is a method for the production of porous inorganic beads with hierarchical pore structures for use, for example, in chromatographic separation procedures. The method comprises combining in a liquid medium at least one porous templating particle and a matrix material precursor under conditions such that the matrix material precursor is allowed to infiltrate the templating particle(s); allowing the matrix material precursor to solidify to form composite beads; and removing the templating particle(s) from the composite bead(s) thereby forming porous beads comprising a porous matrix supplemented with one or more larger pores corresponding to the cavities left by the removed templating particle(s). Preferred are silica, titania and zirconia beads. The invention also provides a separation matrix comprising inorganic beads exhibiting a hierarchical pore structure. The inorganic beads have large surface areas, thereby allowing for high adsorption capacities of target molecules, and improved mass transport properties, particularly at high flow rates.

The present invention relates to a method for the production of porousbeads for use, for example, in a chromatographic separation procedure.In particular, the invention relates to a method for creating inorganicbeads with hierarchical pore structures that give improved masstransport properties, particularly at high flow rates.

A general problem in chromatography, adsorption processes, heterogeneouscatalysis etc, where porous particles are used, is that the masstransport rate is strongly dependent on the particle size. Rapid masstransport can be achieved by decreasing the particle size, but smallparticles will also increase the backpressure of the packed beds. Hence,a compromise must be made between the mass transport rate and thepressure-flow properties. One way to improve the mass transport in aporous particle is to introduce a hierarchical pore structure, wherelarge feeder pores from the particle surface open into a network ofsmaller pores with a large surface area available for adsorption.

There are several published reports describing templating procedures forpreparing porous inorganic oxide structures, suitably by infiltratingthe void spaces of organic or bacterial templates with inorganicprecursors. For example, latex particles (non-porous nanosized beads)have been used as templates to form cavities in porous bulk structures(WO 99/21796 (Antonietti, M. et al; U.S. Pat. No. 5,951,962 (Mueller, U.et al); WO 00/26157, (Yang, P. et al); Antonietti, M. et al, Adv.Mater., (1992), 10(2), 710-712; Wang, D, et al, Adv. Mater., (2001), 13,364-371; Yang, Z., et al, Chinese Science Bulletin, (2001), 45(21),1785-90); Meyer, U. et al, Adv. Mater., (2002), 14(23), 1768-72).Inorganic frameworks composed of oxides of Si, Ti, Zr, Al, W, Fe, Sb,and a Zr/Y mixture have been prepared from metal alkoxide precursorstemplated around polystyrene spheres to form highly ordered macroporousmaterials (Holland, B. T. et al, Chem. Mater., (1999), 11, 795-805).

The fabrication of hierarchically ordered structures has added a newdimension to existing processes for preparing nanostructured materials.Zhang, B. et al (J. Chem. Soc. Chem. Commun., (2000), 781-2) describesthe synthesis and characterisation of hierarchically structured zeolitefibres containing ordered pores at the nano- and micro-scopic lengthscale using an aqueous dispersion of zeolite nanoparticles as buildingblocks. Fan, H. et al, (J. Non-Cryst. Solids, (2001), 285, 71-78)describe the synthesis of nanoporous silica particles having welldefined pore structures and voids through an aerosol-assisted selfassembly process templated by surfactant, polystyrene spheres and oil-inwater emulsions. In this process, the polystyrene spheres serve astemplates for isolated cavities rather than by infiltrating porousbeads.

The various methods suggested in the prior art suffer from certaindrawbacks and limitations. These include, poor connectivities of thepore structures: whenever superpores are produced by templating withparticles dispersed in the porous matrix, the connectivity of the cavitynetwork is dependent on extensive particle-particle contacts that may bedifficult to achieve. For example, using zeolites as building blocksmeans that the small pores of porous structured materials will be ofsub-nanometer dimensions, which is too small for their use aschromatography media for biomolecules. There are practical difficultiesapplying the above techniques for the preparation of hierarchicallystructured porous beads (in particular monodispersed beads which aresuitable for such applications as chromatography, catalysis and solidphase synthesis). Thus, there is still a need for improved methods forthe production of hierarchically structured porous inorganic beads.

Accordingly, one object of the present invention is to provide a porousinorganic bead, whereby an essentially hierarchical network of pores isprovided in each bead, for use, for example in chromatographicseparation procedures, catalysis, solid phase synthesis, etc. It hasbeen surprisingly found that, through the use of large-pore, poroustemplating particles, porous inorganic beads may be produced wherein thebeads exhibit a hierarchical pore structure that favours improved masstransport in process chromatography media. Beads having a hierarchicalpore structure may also be obtained using porous templating particles inwhich the templating process is performed in the presence of asurfactant. The resultant beads have large surface areas, therebyallowing for high adsorption capacities of target molecules. This isachieved by the methods of producing porous inorganic beads as definedin the appended claims.

In a first aspect of the present invention, there is provided a methodof producing at least one porous bead, which comprises the steps of:

i) combining in a liquid medium at least one porous templating particleand a matrix material precursor under conditions such that said matrixmaterial precursor is allowed to infiltrate said templating particle(s);

ii) allowing said matrix material precursor to solidify to formcomposite beads; and

iii) removing said templating particle(s) from said composite bead(s)thereby forming porous beads comprising a porous matrix supplementedwith one or more larger pores corresponding to the cavities left by theremoved templating particle(s);

characterised in that said at least one porous templating particle is abead having a macroporous structure and/or said combining step i) isperformed in the presence of a surfactant;

whereby an essentially hierarchical network of pores is provided in eachbead.

In one preferred embodiment of the method, the porous templatingparticle is a bead having a macroporous structure and the combining stepi) is performed in the absence of a surfactant. In a second preferredembodiment, the porous templating particle is a bead having amacroporous structure and said combining step i) is performed in thepresence of a surfactant.

In the context of the present invention, the term “templating” means thesolidification of a fluid in the presence of solid features and where,upon removal of said solid features a corresponding cavity is formed.The term “hierarchical” is used to describe a porous structure withlarge pores open to the exterior. On the walls of these large pores, asystem of smaller pores opens. Optionally, a further system of evensmaller pores may open on the walls of these pores etc. A hierarchicalpore structure may also have a fractal character, in which case thelarger pores continuously divide into smaller pores. The term“multimodal” when referring to pore structure, means a structure havingseveral distinct classes of pore sizes. Typically, pores may be“bimodal” or “trimodal”, having respectively two or three distinctclasses of pore size. These may or may not be connected in ahierarchical structure. By the term “intraparticle pore volume”, it ismeant the volume of pores inside the beads, as opposed to theinterstitial pores in a packed bed of beads. According to the IUPACdefinition, “micropores” are of a diameter <2 nm, “mesopores” have adiameter within the interval of 2-50 nm and “macropores” are of adiameter >50 nm.

Accordingly, the method of the present invention provides porousinorganic beads having a multimodal hierarchical pore structure.Typically, the method produces beads having a bimodal or sometimestrimodal, preferably bimodal hierarchical pore structure, with smallerpores, usually the size of the type conventionally denoted as mesopores,being connected to macroporous cavities or channels that are formed whenthe templating particle is removed. The said cavities or channels aresometimes for reasons of simplicity referred to herein as “superpores”,and are usually of diameter in the range of between about 50 nm andabout 20,000 nm. The conventional mesopores are for the same reasonsometimes denoted “small pores”, even though the term “small pores” asused herein may also include micropores. Accordingly, the small poresare of smaller diameter than superpores and may have a diameter in therange of between about 1 nm and about 100 nm.

The overall shape of the porous inorganic beads according to the presentinvention will depend generally on the shape of the templating particlefrom which the beads are derived. Preferably, beads prepared by themethod of the invention are spherical or substantially spherical inshape, having a diameter in the range from about 1 μm to about 500 μm.In preferred embodiments, the bead size is in the range from about 10 μmto about 100 μm, and in particularly preferred embodiments from about 30μm to 60 μm.

Matrix material precursors suitable for use in the method according tothe present invention are suitably metal salts or complexes, ormetallo-organic compounds, which are capable of being converted intogels, and wherein the gels can be converted to the corresponding metaloxide, or alternatively converted to the corresponding nitride, borideor carbide. Preferably, the matrix material precursor is a metal salt ora metal or metalloid complex. Preferably, the precursor is soluble in asuitable reaction medium. Suitable metal salts include C₁-C₄ metalalkoxides, carbonates and carboxylates of metals and metalloids such assilicon, titanium, zirconium, tungsten, calcium, aluminum and boron.Suitable complexes include acetoacetonates, carbonyls andcyclopentadienyls of metals and metalloids, such as silicon, titanium,zirconium, tungsten, cerium, hafnium, iron, aluminium, calcium andboron. Alternatively, the precursor may be present as a suspension, forexample, in the form of a colloidal sol of silica, titania or zirconia.

In one preferred embodiment, the matrix material precursor istetra-C₁-C₄ alkyl orthosilicate which, upon calcination, forms SiO₂(silica) porous beads. Suitable tetraalkyl orthosilicates includetetramethyl orthosilicate and tetraethyl orthosilicate. In anotherpreferred embodiment, the matrix material precursor is titanium(IV)isopropoxide which, upon calcination, forms TiO₂ (titania) porous beads.In a still further preferred embodiment, the matrix material precursoris a zirconium(IV) propoxide which, upon calcination, forms ZrO₂(zirconia) porous beads.

The porous inorganic beads according to the present invention may beproduced according to the “sol-gel” method, wherein macroporous(large-pore) template particles are infiltrated with a sol-gel matrixmaterial precursor solution, suitably an aqueous or aqueous/C₁-C₄alcohol solution. The matrix material precursor undergoes hydrolysis andcondensation to form the corresponding metal oxide within the porestructure of the templating particle. It is important to keep the amountof precursor to a minimum in order to prevent the formation of excessinorganic material on the surface of the beads and reduce the effect of“clumping”. Thus, the precursor solution only fills the templatingparticles such that, following gelation, a free-flowing powder ofcomposite beads is formed. The composite beads are then calcined inorder to remove template particle skeletons, thereby resulting in theformation of meso-macroporous beads having a hierarchical porestructure. Without being bound by theory, the meso pores in theresultant bead structure are likely to be formed from interstitial poresformed during the gelation of the sol-gel matrix material precursor.Some shrinkage occurs in the inorganic bead after the calcination step,particularly in the case of titania and zirconia bead types.

Suitably, for preparing beads having a hierarchical structure, theporous templating particle is a bead having a macroporous (i.e. largepore) structure with at least 50% of the pore volume in pores withdiameters in the range from about 40 nm to 20,000 nm, preferably fromabout 100 nm to 10,000 nm. In one embodiment, the porous beads areorganic beads, suitably styrene-divinylbenzene beads such as SOURCE 30RPC, SOURCE 30 Q, SOURCE 30 S, SOURCE 15 ETH, SOURCE 15 RPC, SOURCE 15S, SOURCE 15 Q (all functionalised polydivinylbenzene beads fromAmersham Biosciences), F-BAS/18, F-BAS/26 (both large-porepolydivinylbenzene bead prototypes from Dyno Speciality Polymers),large-pore glycidyl methacrylate/glycerol dimethacrylate (GMA/GDMA),styrene/ethylene glycol dimethacrylate beads, foamball beads (e.g.prepared according to U.S. Pat. No. 5,902,834, Porrvik, I.). Othersuitable templating particles are large-pore beads prepared fromphenol-formaldehyde resins, melamine-formaldehyde resins, polyolefins,cellulose esters, vinyl polymers, polyurethanes, carbon beads, etc. Suchtemplating beads are stable over the range from pH 2 to 12, therebymaking possible the use of acidic or basic conditions during thetemplating process.

In another embodiment, an inorganic templating particle may be used,suitably silica, diatomite, glass, alumina or silicate mineral particlesthat can afterwards be dissolved by leaching with e.g. alkali orhydrofluoric acid.

In a preferred embodiment, the templating particles are selected fromporous organic polymer beads having at least 50% of their totalintraparticle pore volume in pores in the range from 100 nm to 10,000 nmpore diameter, preferably from 200 nm to 10,000 nm, most preferably from250 nm to 10,000 nm.

The surface chemistry of the templating particle is an important factorin the outcome of the method of the present invention. Thus, the surfaceproperties of the templating particle, in terms of surfacehydrophilicity or hydrophobicity, should be controlled in an appropriateway, such that the templating particles are preferably wetted by thematrix material precursor solution, thereby enhancing infiltration ofthe templating particle by the matrix material precursor. Accordingly,in an advantageous embodiment, the present method further comprises thestep of treating the templating particles with a surface-modifyingagent. Such treatment of the templating particles may be before, orduring, the addition thereof to the templating mixture. Treatment beforestep i) is to be particularly preferred. The terms “treated” and“treatment” are understood to mean herein that the surface modifyingagent will react with or adsorb to the particle surface to a sufficientextent to provide the desirable surface characteristics.

For preparing silica beads according to the present invention, thepreferred surface characteristics of the template particles arehydrophillic in nature. The template particle surface may be modified bymeans of an agent in order to enhance the hydrophillicity of thesurface. In functional terms, the said agent may be any molecule withone or more hydrophillic regions or groups, such a molecule beingcapable of reacting with, or physically adsorbing to the particlesurface such that the hydrophillic regions or groups point outwards fromthe surface. Suitably, surface modification includes chemical means,such as by derivatisation of the template particle with sulphonate (—SO₃⁻), or amino (—NH₂) groups, or by sorbitol functionalisation.Alternatively, surface treatment of the templating particle may beperformed by adsorbing surface modifying moieties such aspoly(diallyldimethylammonium chloride) and phenyldextran. Preferredbeads for templating silica beads are selected from hydrophillic beads,e.g. sulfonated or aminated styrene-divinylbenzene beads, andamine-derivatised GMA/GDMA beads.

Alternatively, for preparing titania beads and zirconia beads accordingto the invention, the surface characteristics of the templatingparticles are preferably hydrophobic in nature. Preferred beads areselected from F-BAS/18, F-BAS/26, EGDMA/styrene and styrene foamballs.The surface of the templating particle may be modified to furtherenhance hydrophobicity of the surface by treatment with a hydrophobicagent such as a fatty acid, a fatty amine, a hydrophobic silane, etc. Infunctional terms, said agent can be any molecule with one or morehydrophobic regions or groups that is capable of reacting with orphysically adsorbing onto the surface of the templating particle is sucha manner that the hydrophobic regions or groups point outwards from thesurface. Accordingly, a suitable agent is readily selected by theskilled person, since a large number of such surface modifying agentsare commercially available.

Methods for modification of surfaces are well known to the skilledperson and various handbooks and literature describe the generalprinciples and considerations needed. See, for example, “IonExchangers”, K. Dorfner (Ed.), Berlin (1991).

When porous beads are used as templates according to the presentinvention, the characteristics of their pore surfaces are of importancefor the outcome of the process. In the case where the beads areimpregnated with an aqueous solution containing e.g. a silica precursor,it is essential that the beads are well wetted by the solution (i.e.that they are sufficiently hydrophilic, which can be assessed by a waterabsorption test) and it is also advantageous if the pore surfaces have ahigh affinity to the inorganic material formed (e.g. by having a basiccharacter in the case of silica which is an acidic oxide). In the casewhere the dry beads are impregnated with moisture-sensitive precursorssuch as titanium or zirconium alkoxides, the best results are obtainedif the pore surfaces are hydrophobic.

The liquid medium used for performing the reaction may be any mediumcompatible with handling, and subsequently hydrolysing, the matrixmaterial precursor to form composite beads. For example, with silicaprecursors, water may be employed as the reaction medium. Alternatively,the liquid medium may be a mixture of water with a water-miscibleorganic solubilizer, such as a C₁-C₄ alcohol, for example methanol,ethanol or isopropanol, 1,4-dioxane, tetrahydrofuran, or acetonitrile.The reaction can also be performed in a two-phase liquid reactionmedium, which consists of an aqueous phase and an organic phase which isimmiscible or only slightly miscible with water. In relation to thereaction according to the present invention, the organic phase should beinert. A suitable organic phase includes hydrocarbons (e.g. toluene),ethers (e.g. diethyl ether), ketones (e.g. methylethyl ketone), esters,higher alcohols, etc. Care should be taken when handling certain matrixmaterial precursors, for example, precursors of titania and zirconia,which must be infiltrated under completely water-free conditions.Subsequent hydrolysis of the precursor is carried by the controlledaddition of water or moisture to a non-aqueous solution of theprecursor. The reaction medium or the two-phase mixture is suitablyagitated, in particular stirred, during the reaction.

The formation of a hierarchical pore structure may be enhanced by theaddition of one or more surfactants or other sacrificial pore formingagents to the templating reaction mixture. Thus, in one embodimentaccording to the present invention, the combining step i) is performedin the presence of a surfactant. The surfactant may be present in theliquid medium when the porous templating particle(s) are added therein.Alternatively, the templating particle(s) may be treated with asurfactant before they are added to the liquid medium. Suitablesurfactants include quaternary ammonium salts (e.g.cetyltrimethylammonium bromide, CTAB), sulphosuccinates (e.g. sodiumdioctyl sulphosuccinate), ethoxylated alcohols (e.g. ethoxylated fattyalcohols or alkyl phenols), polyethers (e.g. ethylene oxide-propyleneoxide block copolymers), alkyl sulphates (e.g. sodium dodecyl sulphate),sulphonates (e.g. sodium dodecylbenzene sulphonate), etc. The additionof a surfactant to the templating reaction mixture is particularlyadvantageous if template particles having pore diameters less that about100 nm are employed. For example, hierarchical porous silica beads maybe produced using SOURCE15ETH templating beads (with pores <100 nm) andthe surfactant CTAB.

After the liquid matrix material precursor has been allowed toinfiltrate the pores of the template, the precursor is converted to agel by aqueous hydrolysis of the matrix material precursor. Optionally,the hydrolysis may be performed under acidic or basic conditions,wherein the presence of an acid or a base may facilitate the conversion.Preferably, the matrix material precursor is converted into a gel bytreatment of the precursor with water and (in the case of siliconalkoxides), more preferably by treatment with one or more dilute aqueousacids or bases. When an acidic or a basic medium is used, the acid orbase is present at a concentration range of about 1 mmol/l to 0.5 mol/l,most preferably about 10 mmol/l. Preferred acids include acetic acid,hydrochloric acid, sulphuric acid, nitric acid etc. Preferred basesinclude ammonia, amines, alkali hydroxides etc. Most preferably, whenthe matrix material precursor is a titanium or a zirconium alkoxide,conversion to a gel is effected by water treatment, whereas with siliconalkoxide precursors addition of approximately 10 mmol/l hydrochloricacid is to be preferred.

The gel conversion reaction to form a composite bead is suitablyperformed at a temperature of between 20° C. and 100° C. Generally,higher temperatures within this range will facilitate infiltration ofthe liquid ceramic precursor and conversion to a gel, a preferredtemperature range being between 50° C. and 80° C. The reaction issuitably allowed to proceed from about 1 hour to about 5 days,preferably from 12 hours to 72 hours. The time and temperature needed toconvert the ceramic precursor to a gel will vary due to the reactivityof the precursor used and due to the presence of any accelerating (e.g.acids and bases) or retarding compounds.

The composite beads in which gel is disposed throughout at least aportion of the templating particle, may be converted to the porousinorganic beads according to the present invention, by heating thetemplated gel, suitably at a temperature of between about 300° C. and1000° C. for a period of time sufficient to convert the gel to a ceramicmaterial and concomitant removal of the template. A preferredtemperature suitable for calcining the beads will be in the range from550° C. to about 750° C., depending on the inorganic composition of thegel. It is generally preferred to calcine the beads by means of atwo-stage heating program, wherein, in a first stage the temperature israised from ambient to the final calcining temperature in a gradientover a period of 1.5-2.5 hours. In a second stage, the beads arecalcined at the preferred temperature for a period of between 1 hour to24 hours. It is preferred to pass a constant air flow through thecalcination furnace, so as to ensure the complete combustion and removalof the organic template material.

In the embodiment wherein an inorganic templating particle is employed,the template must be removed from the composite beads by methods that donot affect the matrix material. For example, a porous silica templatingparticle may be used to template titania or zirconia beads. Titania orzirconia matrix material precursor is allowed to infiltrate the pores ofthe silica template and form metal oxide as described. The templatingparticles may be removed from the composite beads by the use ofconventional leaching methods depending on the nature of the bead matrixand the templating particle. Naturally, the leaching agent should beselected so as not to have a negative impact on the properties of thebead, while being sufficiently aggressive for efficient removal. In oneembodiment, removal of the templating particle may be performed byetching the silica template by the addition of fluorine compounds, forexample, hydrofluoric acid or ammonium bifluoride. In anotherembodiment, the etching is performed by the addition of one or morebases, for example sodium or potassium hydroxide, which are suitable inthe case of alkali soluble templating particles.

As mentioned above, in an advantageous embodiment according to thepresent method, the templating particles are surface treated beforecombining the particles with matrix material precursor. Such pre-treatedparticles may be prepared as a step preceding step i). Alternatively,pre-treated particles for use as templates may be obtained fromcommercial sources. For example, large-pore styrene-divinylbenzene beadswith anion exchange functionality (and hence basic and hydrophilic poresurfaces) are available from Purolite International as a productdesignated A501P.

In a typical example of the preparation of silica beads, organictemplate beads, suitably sulfonated or aminated F/BAS-18 or FIBAS-26, oramine derivatised GMA/GDMA beads, are dried in an oven at 60° C.overnight. Dried beads are soaked in 0.01M HCl solution in a reactorequipped with stirring means. By this procedure, fewer aggregates ofbeads are formed. Optionally, a surfactant may be included in thereaction mixture, preferably prior to addition of the beads.Tetramethylorthosilicate is added to the reactor and the reactionallowed to proceed at 70° C. during 72 hours. Either acidic or basicsol-gel hydrolysis may be employed, acidic conditions being moregenerally applicable. The beads are washed on a glass filter withdeionized water and the washed beads dried at 60° C. overnight. Thedried beads are calcined, complete combustion of organic material beingensured by allowing a constant airflow through the oven.

For preparing titania and zirconia beads, templating particles having ahydrophobic surface character are to be preferred. Suitable examplesthat may be used include styrene/DVB templates, F/BAS-18, F/BAS-26,EGDMA/styrene and foamballs. The dried templating particles are soakedin a limited amount of bulk titania (or zirconia) precursor andthereafter, the reaction is initiated by adding distilled water.

In a second aspect of the present invention, there is provided aseparation matrix comprising porous beads produced according to themethod described above, characterised in that an essentiallyhierarchical network of pores is provided in each bead.

Preferably, the beads are spherical or substantially spherical in shape,having a diameter in the range from about 1 μm to about 500 μm,preferably in the range from about 10 μm to about 100 μm.

The porous inorganic beads according to the invention may be used asseparation matrices in process chromatography methods. The inorganicbeads have large surface areas, thereby allowing for high adsorptioncapacities of target molecules, and improved mass transport properties.Thus, in a third embodiment, there is provided a liquid chromatographyprocedure for separating and distinguishing at least one solute in achromatography column containing a stationary separation matrix,including the steps of flowing through said column a discrete volume ofa liquid mixture containing said solute(s) and eluting from saidseparation matrix said solute(s) bound thereto; characterised in thatsaid separation matrix is comprised of porous beads exhibiting anessentially hierarchical pore structure that favours mass transport,wherein the beads are obtainable according to the method of theinvention.

One specific advantage of the method for producing beads according tothe present invention is that it is possible to prepare mono-dispersedbeads, providing that mono-dispersed polymer beads are used as template.The present technique will also produce different types of porestructures, thereby widening the scope of possible structures. Thepresence of a physically connected structure in the porous bead templatemeans that the superpores in the resulting hierarchically structuredbead will be connected throughout the bead. It is also possible tomanipulate the porosity and the mechanical properties by varying theamount of precursor used and the amounts of surfactants or othersacrificial pore forming agents.

In many chromatography applications the pore surfaces of the beads maybe chemically modified to give the desired adsorption selectivity. Themodification can involve the fixation of ligands (interacting groups)including charged groups for ion exchange chromatography (for example,—N⁺(CH₃)₃), —SO₃ ⁻, —COO⁻); hydrophobic groups (hydrophobic interactionchromatography); or groups with affinity to specific proteins etc(affinity chromatography). Alternatively, applications may also involvecomplete coating of the pore surfaces with a hydrophobic layer (reversedphase chromatography) or a protein-inert hydrophilic layer (gelfiltration chromatography). In some cases it may be necessary to combinecoating and ligand immobilisation processes, for example, by firstapplying an inert hydrophilic coating and then attaching the ligands tothe coating.

The invention is further illustrated by reference to the followingexamples and figures which are presented herein for illustrativepurposes only and should not be construed as limiting the scope of thepresent invention as defined by the appended claims.

FIG. 1 shows Scanning Electron Microscope (SEM) pictures of silica beadsof Example 1 in low (A) and high (B) magnifications. These beads show ahierarchical structure with a network of superpores (diameter up to 5microns) and a set of small pores in the struts of the superporestructure.

FIG. 2 shows SEM pictures of silica beads of Example 2 in low (A) andhigh (B) magnifications. These beads show a hierarchical structure witha network of superpores (diameter up to 10 microns) and a set of smallpores in the struts of the superpore structure.

FIG. 3 shows SEM pictures of titania beads of Example 3 in low (A) andhigh (B) magnifications. These beads show a hierarchical structure witha network of micron-sized superpores connected to a set of small pores.

FIG. 4 is a SEM picture of the interior structure of a titania beadaccording to Example 4 in high magnification. These beads show ahierarchical structure with a network of micron-sized superporesconnected to a set of small pores.

FIG. 5 shows SEM pictures of zirconia beads of Example 5 in low (A) andhigh (B) magnification. These beads show a hierarchical structure with anetwork of micron-sized superpores connected to a set of small pores.

FIG. 6 shows SEM pictures of zirconia beads of Example 6 in low (A) andhigh (B) magnification. These beads show a hierarchical structure with anetwork of micron-sized superpores connected to a set of small pores.

FIG. 7 is a SEM picture showing a zirconia bead of Example 7 in highmagnification. These beads show a hierarchical structure with a networkof micron-sized superpores connected to a set of small pores.

FIG. 8 shows SEM pictures of a comparative example of the preparation ofnon-hierarchical silica beads (U544081, Silica) according to Example 8in low (A) and high (B) magnification. These beads show a typicalsol-gel pore structure without signs of hierarchy.

FIG. 9 is a SEM picture of a comparative example of the preparation oftitania beads (U544068, Titania) according to Example 9 showing atypical sol-gel pore structure without signs of hierarchy.

FIG. 10 shows SEM pictures of silica beads of Example 10 in low (A) andhigh (B) magnifications. These beads show a pore structure having ahierarchical pore structure with 100-200 nm superpores connected to aset of smaller pores.

FIG. 11 is a plot showing peak width for 80 kD dextran eluted on acolumn of hierarchical silica beads (U544083) prepared according toExample 10. The absence of any width increase above 0.5 ml/min is a signof improved mass transport from the hierarchical structure.

EXAMPLES Example 1 Preparation of Silica Beads (U544077, Silica)

Foamballs (average particle size 150 microns, pore size 5-10 microns)were prepared from styrene and divinylbenzene according to U.S. Pat. No.5,902,834, Example 1.

Dried foamballs (2 g) were swollen in dichloroethane for 1 hour.Concentrated sulphuric acid (60 ml) was added and the reaction wasallowed to proceed at 70° C. overnight. The sulphuric acid was removedfrom the beads by suction on a glass filter and the beads washed on aglass filter with plenty of water.

The sulphonated template beads were dried in an oven at 60° C.overnight. Dried beads (0.1 g) were soaked in 0.01M hydrochloric acid(10 ml) in a reactor equipped with an anchor stirrer.Tetramethylorthosilicate (135 μl) was added and the reaction was allowedto proceed at 70° C. for three days. The beads were washed on a glassfilter with plenty of deionized water and subsequently dried at 60° C.overnight. The dried beads were calcined according to the followingprogram; i) 0-90 min: 25-550° C. ii) 90-1140 min: 550° C.

After 3.5-4 hours a constant airflow was allowed through the furnace toguarantee complete combustion of organic material.

Example 2 Preparation of Silica Beads (U544078, Silica)

Foamballs (average particle size 150 microns, pore size 5-10 microns)were prepared from styrene and divinylbenzene as in Example 1 above.

Dried foamballs (2 g) were swollen in dichloroethane for 1 hour.Concentrated sulphuric acid (60 ml) was added and the reaction wasallowed to proceed at 70° C. overnight. The sulphuric acid was removedfrom the beads by suction on a glass filter. The beads were washed onglass filter with plenty of water.

Poly(diallyl dimethyl ammonium chloride) (polyDADMAC; 2.5 ml) wasdiluted to 10 ml with distilled water. Sulphonated organic beads weredried by suction on a glass filter. Thereafter, the organic beads 1.5 g)were swollen in the polymer solution in a reactor equipped with ananchor stirrer at room temperature for 6.5 hours. The beads were washedon a glass filter with plenty of distilled water.

The polyDADMAC-coated template beads were dried in an oven at 70° C.overnight. Dried beads (0.5 g) were soaked in 0.01M hydrochloric acid(10 ml) in a reactor equipped with an anchor stirrer.Tetramethylorthosilicate (6.8 ml) was added and the reaction was allowedto proceed at 70° C. for three days. The beads were washed on a glassfilter with plenty of deionized water and dried at 60° C. overnight. Thedried beads were calcined according to the following program; i) 0-90min: 25-550° C. ii) 90-1140 min: 550° C.

After 3.5-4 hours a constant airflow was allowed through the furnace toguarantee complete combustion of organic material.

Example 3 Preparation of Titania Beads (U544084c, Titania)

78 micron monodisperse polydivinylbenzene beads (total pore volume 2.1ml/g, <10 nm pore diameter 0.027 ml/g, 10-40 nm 0.052 ml/g, 40-100 nm0.135 ml/g, 100-200 nm 0.285 ml/g, 200-400 nm 1.050 ml/g, 400-1000 nm0.534 ml/g) were obtained from Dyno Particles A/S, Lillestroem Norway(sample designation Dynospheres EXP-PD-70-RXE F-BAS/26-F).

Dried beads (4.0 g) were weighed into a flask and were thereafteradditionally dried at 60° C. overnight. Titanium(IV)isopropoxide (2 g)was added and infiltration into the beads was allowed to proceedovernight. Water (100 ml) was then added and the reaction was allowed toproceed at room temperature for 4-7 hours on an agitation table. Theinorganic/organic hybrid beads were washed on a glass filter with 3portions of deionized water. The hybrid beads were then dried in an ovenat 60° C. overnight. The calcination of titania beads followed theprogram of the silica beads described above.

Example 4 Preparation of Titania Beads (U544084a, Titania)

55 micron monodisperse polydivinylbenzene beads (total pore volume 2.0ml/g, <10 nm pore diameter 0.017 ml/g, 10-40 nm 0.023 ml/g, 40-100 nm0.139 ml/g, 100-200 nm 0.346 ml/g, 200-400 nm 0.932 ml/g, 400-1000 nm0.536 ml/g) were obtained from Dyno Particles A/S, Lillestroem Norway(sample designation Dynospheres EXP-PD-70-RXE F-BAS/18-F).

Dried beads (4.0 g) were weighed into a flask and were thereafteradditionally dried at 60° C. overnight. Titanium(IV)isopropoxide (2 g)was added and infiltration into the beads was allowed to proceedovernight. Water (100 ml) was then added and the reaction was allowed toproceed at room temperature for 4-7 hours on an agitation table. Theinorganic/organic hybrid beads were washed on a glass filter with 3portions of deionized water. The hybrid beads were dried in an oven at60° C. overnight. Calcination of titania beads followed the program ofthe silica beads described above.

Example 5 Preparation of Zirconia Beads (U515057, Zirconia)

78 micron monodisperse polydivinylbenzene beads (total pore volume 2.1ml/g, <10 nm pore diameter 0.027 ml/g, 10-40 nm 0.052 ml/g, 40-100 nm0.135 ml/g, 100-200 nm 0.285 ml/g, 200-400 nm 1.050 ml/g, 400-1000 nm0.534 ml/g) were obtained from Dyno Particles A/S, Lillestroem Norway(sample designation Dynospheres EXP-PD-70-RXE F-BAS/26-F).

Dried beads (4.0 g) were weighed into a flask and were thereafteradditionally dried at 60° C. overnight. Zirconium(IV)propoxide (2 ml)was added and the infiltration into the beads was allowed to proceedovernight. Water (100 ml) was then added and the reaction was allowed toproceed at room temperature for 4-7 hours on an agitation table. Theinorganic/organic hybrid beads were washed on a glass filter with 3portions of deionized water. The hybrid beads were dried in an oven at60° C. overnight. The calcination of zirconia beads followed the programbelow. i) 0-120 min: 25-750° C. ii) 120-720 min: 750° C.

After 3.5-4 hours a constant airflow was allowed through the oven toguarantee complete combustion of organic material. The zirconia beadshad a surface area (BET N₂ adsorption) of 21.6 m²/g.

Example 6 Preparation of Zirconia Beads (U515050, Zirconia)

Ethylene glycol dimethacrylate (42.3 g), 2-ethyl hexanol (119.0 g),styrene (8.9 g) and azobisdimethylvaleronitrile (1.0 g) were mixed in abeaker. This solution was poured into a 1 l glass reactor fitted with ananchor stirrer, containing 1% aqueous polyvinyl alcohol solution (Mowiol40-88, Clariant) (680 ml) and potassium dichromate (3.6 g). The phaseswere emulsified at 450 rpm and the temperature was raised to 650C. After2 hour, 1 M NaOH (50 ml) was added and the polymerisation was allowed toproceed overnight at 450 rpm. The resulting beads were washed with hotwater, 20% ethanol, 50% ethanol and 100% ethanol. They were then sievedbetween 40 and 100 μm and dried at 60° C. The beads had a coarse porestructure with pores up to 1 μm diameter, as judged from SEM pictures.

Dried beads (5.3 g) were weighed into a flask and were thereafteradditionally dried at 50° C. overnight. Zirconium(IV)propoxide (2.6 ml)was added and infiltration into the beads was allowed to proceedovernight. Water (100 ml) was then added and the reaction was allowed toproceed at room temperature for 4-7 hours on an agitation table. Theinorganic/organic hybrid beads were washed on a glass filter with 3portions of deionized water. The hybrid beads were dried in an oven at60° C. overnight. The calcination of zirconia beads followed the programbelow. i) 0-120 min: 25-750° C. ii) 120-720 min: 750° C.

After 3.5-4 hours, a constant airflow was allowed through the oven toguarantee complete combustion of organic material. The zirconia beadshad a surface area (BET N₂ adsorption) of 19.9 m²/g.

Example 7 Preparation of Zirconia Beads (U515042d, Zirconia)

Ethylene glycol dimethacrylate (42.3 g), 2-ethyl hexanol (119.0 g),styrene (8.9 g) and azobisdimethylvaleronitrile (1.0 g) were mixed in abeaker. This solution was poured into a 1 l glass reactor fitted with ananchor stirrer, containing 1% aqueous polyvinyl alcohol solution (Mowiol40-88, Clariant) (680 ml) and potassium dichromate (3.6 g). The phaseswere emulsified at 450 rpm and the temperature was raised to 65° C.After 2 h, 1 M NaOH (50 ml) was added and the polymerisation was allowedto proceed overnight at 450 rpm. The resulting beads were washed withhot water, 20% ethanol, 50% ethanol and 100% ethanol. They were thensieved between 40 and 100 μm and dried at 60° C. The beads had a coarsepore structure with pores up to 1 μm diameter, as judged from SEMpictures.

Dried beads, 30 mg, were weighed into a flask and were thereafteradditionally dried at 60° C. overnight. Zirconium(IV)propoxide (0.1 ml)was added and infiltration into the beads was allowed to proceedovernight. Water (100 ml) was then added and the reaction was allowed toproceed at room temperature for 4-7 hours on an agitation table. Theinorganic/organic hybrid beads were washed on a glass filter with 3portions of deionized water. The hybrid beads were dried in an oven at60° C. overnight. The calcination of zirconia beads followed the programbelow. i) 0-120 min: 25-750° C. ii) 120-720 min: 750° C.

After 3.5-4 hours a constant airflow was allowed through the oven toguarantee complete combustion of organic material.

Example 8 Comparative Example: Preparation of Non-Hierarchical SilicaBeads (U544081, Silica)

15 μm monodisperse SOURCE 15ETH beads (Amersham Biosciences, total porevolume 1.1 ml/g, <10 nm pore diameter 0.00 ml/g, 10-40 nm 0.25 ml/g,40-60 nm 0.38 ml/g, 60-100 nm 0.20 ml/g, 100-400 nm 0.24 ml/g, 400-1000nm 0.05 ml/g) were dried in an oven at 60° C. overnight Dried beads (5.0g) were soaked in 0.01M hydrochloric acid (500 ml) in a reactor equippedwith an anchor stirrer. Tetramethylorthosilicate (6.8 ml) was added(without any surfactant addition) and the reaction was allowed toproceed at 70° C. for three days. The beads were washed on a glassfilter with plenty of deionized water and subsequently dried at 60° C.overnight. The dried beads were calcined according to the followingprogram; i) 0-90 min: 25-550° C. ii) 90-690 min: 550° C.

After 3.5-4 hours a constant airflow was allowed through the furnace toguarantee complete combustion of organic material.

FIG. 8 shows SEM pictures of silica beads of the comparative example inlow (A) and high (B) magnifications. These beads show a typical sol-gelpore structure without signs of hierarchy.

Example 9 Comparative Example: Preparation of Titania Beads (U544068,Titania)

15 micron monodisperse polydivinylbenzene beads (total pore volume 2.1ml/g, <10 nm pore diameter 0.427 ml/g, 10-40 nm 0.297 ml/g, 40-100 nm0.378 ml/g, 100-200 nm 0.337 ml/g, 200-400 nm 0.333 ml/g, 400-1000 nm0.368 ml/g) were obtained from Dyno Particles A/S, Lillestroem Norway(sample designation Dynospheres PD15RXE BAS-150).

Dried beads (4.0 g) were weighed into a flask and were thereafteradditionally dried at 60° C. overnight. Titanium(IV)isopropoxide (4 g)was added (without any surfactant addition) and infiltration into thebeads was allowed to proceed overnight. Water (100 ml) was then addedand the reaction was allowed to proceed at room temperature for 4-7hours on an agitation table. The inorganic/organic hybrid beads werewashed on a glass filter with 3 portions of deionized water. The hybridbeads were then dried in an oven at 60° C. overnight. The calcination oftitania beads followed the program of the silica beads described above.

FIG. 9 is a SEM picture of the titania beads of Example 9 showing atypical sol-gel pore structure without signs of hierarchy.

Example 10 Preparation of Silica Beads (U544083, Silica)

15 μm monodisperse SOURCE 30Q beads (Amersham Biosciences, total porevolume 1.1 ml/g, <10 nm pore diameter 0.00 ml/g, 10-40 nm 0.25 ml/g,40-60 nm 0.38 ml/g, 60-100 nm 0.20 ml/g, 100-400 nm 0.24 ml/g, 400-1000nm 0.05 ml/g) were dried in an oven at 60° C. overnight. Dried beads(5.0 g) were soaked in 500 ml 0.01M hydrochloric acid+10 g cetyltrimethylammonium bromide (surfactant) in a reactor equipped with ananchor stirrer. Tetramethylorthosilicate (6.8 ml) was added and thereaction was allowed to proceed at 70° C. for three days. The beads werewashed on a glass filter with plenty of deionized water and subsequentlydried at 60° C. overnight. The dried beads were calcined according tothe following program; i) 0-90 min: 25-550° C. ii) 90-690 min: 550° C.

After 3.5-4 hours a constant airflow was allowed through the furnace toguarantee complete combustion of organic material. The calcined beadshad a surface area of 490 m²/g, with a nitrogen adsorption isothermindicating a bimodal pore size distribution.

FIG. 10 shows SEM pictures of silica beads of Example 10 in low (A) andhigh (B) magnifications. These beads show a hierarchical structure with100-200 nm superpores connected to a set of smaller pores.

The beads were then packed into a 5 mm diameter HR 5/5 column (AmershamBiosciences) to a bed height of 4.7 cm and an 80 kD dextran sample wasinjected and eluted isocratically with water. The peak width (calculatedas the second moment of the peak) is plotted versus the volumetric flowrate in FIG. 11. For a normal pore structure, the peak width willincrease monotonically with the flow rate. If the beads have ahierarchical pore structure with superpores penetrating the entire bead,it is expected that the peak width will be constant above a certain flowrate. As FIG. 11 shows, the peak width only increases up to 0.5 ml/minflow rate and then it decreases. This is a sign of an improved masstransport rate, attributed to the hierarchical pore structure.

FIG. 11. Peak width for 80 kD dextran eluted on a column of hierarchicalsilica beads (U544083) prepared according to Example 10. The absence ofany width increase above 0.5 ml/min is a sign of improved mass transportfrom the hierarchical structure.

Example 11 Conversion of Silica Beads into Cation Exchange Beads:Determination of the Dynamic Protein Capacity of the Beads

Organic template beads (SOURCE 15ETH) (20 g) were dried in an oven at65° C. for approx 36 h. Cetyltrimethylammonium bromide (CTAB) (40.4 g)was dissolved in 2000 ml 0.01M HCl solution. To dissolve the surfactantthe solution had to be heated to 45° C. and then cooled to roomtemperature. The dried beads were soaked in the CTAB/HCl solution in areactor equipped with small Teflon blade stirrer for approx. 1.5 h.Tetramethylorthosilicate (27.2 ml) was added and the reaction wasallowed to proceed at 70° C. during 72 hours. The beads were washed on aglass filter with approx. 10 liters of deionized water and the washedbeads dried overnight at 60° C.

The dried beads were calcined in an oven according to the followingprogram: i) 0-90 min: 25-550° C. ii) 90-690 min: 550° C.

After 3.5-4 hours a small constant airflow was allowed through the ovento guarantee complete combustion of organic material. The result waswhite spherical particles having a diameter of approximately 12 micron;total weight of 4.7 g.

Toluene (60 ml) was added to a 100 ml reactor equipped with small Teflonblade stirrer. 4.57 g dried silica beads and 600 microliters deionizedwater were added under stirring. The solution was stirred for approx. 15minutes at room temperature and then glycidoxypropyltrimethoxysilane(10.5 ml) was added to the reactor. The solution was heated to 70° C.and the reaction was allowed to proceed at 70° C. during 22 hours. Thebeads were washed on a glass filter with toluene, ethanol and deionizedwater. Remaining water was removed by dry suction of the beads.

The epoxy activated beads were transferred to a 100 ml vial. Sodiumsulphite (15 g) was dissolved in deionized water (75 ml) and added tothe vial. The solution was slightly basic, about pH 8. The vial wasclosed but an injection needle was inserted through the gasket toprevent high pressure build-up inside the vial. The vial was placed inan oven and the reaction was allowed to proceed at 100° C. during 47hours. The particles were washed with deionized water. Fines wereremoved by elutriating the solution twice in a 50 ml graduated measuringcylinder. The beads were sieved with a 40 micron sieve. The volume ofthe sieved S-Silica was estimated to about 19 ml.

The beads were packed in a HR5/5 column (Amersham Biosciences) andtested with respect to dynamic capacity of the protein chymotrypsinogen.At 300 cm/h flow velocity, the capacity (10% breakthrough level) was 155mg protein/ml gel. At 900 cm/h, the capacity was 160 mg/ml. For acomparable commercial 10 micron cation exchanger, Mono S (AmershamBiosciences), the capacity was 75 mg/ml at 300 cm/h and 74 mg/ml at 900cm/h.

1. A method of producing at least one porous bead, comprising the stepsof: i) combining in a liquid medium at least one porous templatingparticle and a matrix material precursor under conditions such that saidmatrix material precursor infiltrates said templating particle(s); ii)allowing said matrix material precursor to solidify to form compositebeads; and iii) removing said templating particle(s) from said compositebead(s) thereby resulting in porous beads comprising a porous matrixsupplemented with one or more larger pores corresponding to the cavitiesleft by the removed templating particle(s); wherein said at least oneporous templating particle is a bead having a macroporous structure andsaid combining step i) is performed in the presence of a surfactant;whereby a hierarchical network of pores is provided in each bead.
 2. Themethod of claim 1, wherein a surfactant is present in the liquid mediumwhen the templating particle(s) are added therein.
 3. The method ofclaim 1, wherein said templating particle(s) are treated with asurfactant before they are added to the liquid medium.
 4. The method ofclaim 1, further comprising the step of treating the templatingparticles with a surface modifying agent.
 5. The method of claim 4,wherein said treatment step precedes step i).
 6. The method of claim 1,wherein said templating particle(s) are beads having a macroporousstructure with at least 50% of the pore volume in pores with diametersin the range from 40 nm to 20,000 nm.
 7. The method of claim 1, whereinsaid templating particle(s) are selected from porous organic polymerbeads having at least 50% of their total intraparticle pore volume inpores in the range from 100 nm to 10,000 nm pore diameter.
 8. The methodof claim 1, wherein said matrix material precursor is a metal salt or ametal or metalloid complex.
 9. The method of claim 1, wherein saidmatrix material precursor upon calcination forms an oxide selected fromthe group consisting of TiO₂, SiO₂ and ZrO₂.
 10. The method of claim 1,wherein said hierarchical network of pores comprises a bimodalhierarchical pore structure having mesopores connected to macroporouscavities or channels that are formed when the templating particle isremoved.
 11. The method of claim 10, wherein said cavities or channelshave a diameter range of between about 50 nm and about 20,000 nm. 12.The method of claim 10, wherein said cavities or channels have adiameter range of between about 50 nm and about 10,000 nm.
 13. Themethod of claim 10, wherein said mesopores have a diameter range ofbetween about 1 nm and about 100 nm.
 14. A separation matrix comprisingporous beads of claim 1, wherein a hierarchical network of pores isprovided in each bead.
 15. The separation matrix of claim 14, whereinsaid beads are spherical or substantially spherical in shape, having adiameter in the range from about 1 μm to about 500 μm.
 16. Theseparation matrix of claim 14, wherein said beads are spherical orsubstantially spherical in shape having a diameter in the range of 10 μmto about 100 μm.
 17. A liquid chromatography procedure for separatingand distinguishing at least one solute in a chromatography columncontaining a stationary separation matrix, including the steps offlowing through said column a discrete volume of a liquid mixturecontaining said solute(s) and eluting from said separation matrix saidsolute(s) bound thereto; wherein said separation matrix is comprised ofporous beads exhibiting an essentially hierarchical pore structure thatfavours mass transport wherein said beads are obtainable by the methodof claim 1.